Alcoholic fermentation represents the largest application of the yeast Saccahromyces cerevisiae in the field of industrial biotechnology with the production of over 80 billion liters of fuel, industrial and beverage ethanol in 2011. Over the past decade and due to economic and environmental reasons, the world has experienced exponential growth in the production of fuel ethanol (Schubert, 2006). Though lignocellulose is considered to be one of the most promising feedstocks for production of fuel ethanol, the current industrial production of fuel ethanol relies heavily on the fermentation of traditional feedstocks such as sucrose (derived primarily from sugarcane or sweet sugar beets) and glucose obtained from starchy materials (corn, wheat, barley, potatoes etc). The most common organism currently used for bioethanol production is the baker's yeast, Saccharomyces cerevisiae. This yeast catabolizes glucose via the glycolytic Embeden Meyerhof Pathway (EMP) pathway yielding 2 moles ATP per mole of consumed glucose. The efficiency of this pathway in yeast is low with a maximal biomass yield of around 7% and an ethanol yield in the range 90 and 93% of the theoretical value (Ingledew, 1999). However, at industrial scale the 7% of the sugar that is converted to cell mass represents a huge amount of by-product, which though valuable as animal feed significantly lowers the overall yield of the target product, ethanol. Even a slight improvement in ethanol yield by S. cerevisiae, can add several millions 1 liters of ethanol to the worldwide production of ethanol production annually.
In contrast to S. cerevisiae, the bacterium Zymomonas mobilis ferments glucose through Entner Doudorf (ED) pathway. This pathway gives only 1 mole of ATP per mole of glucose, and directs only 3% of glucose to cell biomass achieving ethanol yield of up to 97% of the possible theoretical value (Sprenger, 1996). This indicates that lowering the level of ATP yield during alcoholic fermentation increases ethanol yield with reduced substrate conversion to cell mass. Furthermore, Z. mobilis has another important advantage over S. cerevisiae: in faster fermentation of glucose to ethanol (Sprenger, 1996, Panesar et al., 2006). The higher productivity of Z. mobilis is frequently attributed to faster rate of sugar uptake and subsequent catabolism rather than low ATP yield. Attempts to substitute S. cerevisiae by Z. mobilis for the production of industrial ethanol were considered to increase ethanol yield by 3-4% thereby adding several hundred million liters worldwide annually. However, Z. mobilis has several serious drawbacks which hamper its industrial use and these consist of: (i) a very narrow substrate range (sucrose is hardly fermented with low yield), (ii) natural auxotrophy for lysine, methionine and some vitamins, (iii) non-GRAS status, which prevents use of biomass as feed additive, (iv) requirement for a higher pH to grow (Jeffries, 2005; Bai et al., 2008; Abbas, unpublished finding). Furthermore, the technology of yeast cell utilization for alcoholic fermentation is well developed whereas the use of bacterial cells for ethanol production is far less common. Thus, a better approach to increase ethanol yield and reduce cell mass production is to construct yeast strains which yield less ATP during alcoholic fermentation (e.g. one mole ATP, as Z. mobilis does in ED pathway). These new yeast strains would combine all of the possible advantages of yeast with the high ethanol yield of Z. mobilis. There are several approaches that can be used to achieve this goal, for example: substitution of EMP pathway in yeast by ED pathway from Z. mobilis or other bacteria possessing genes of the pathway; increasing the activity of enzymes involved in generation of futile cycles; construction of recombinant strains with elevated ATPase activity; and the introduction of heterologous genes encoding for plasma membrane symporters.
The first approach tried was to express ED dehydratase and ED aldolase genes edd and eda in a phosphofructokinase deficient mutant of S. cerevisiae (Lancashire et al., 1998). The yeast transformants obtained grew and fermented glucose to ethanol, though activities of ED dehydratase and ED aldolase were not measured. The work described in this patented work, was not further developed as there is no additional reports in the scientific literature. Quite often prokaryotic enzymes display low or no activity in S. cerevisiae hosts (Hahn-Hagerdal et al., 2007). This is probably due in part to improper folding or instability of the expressed bacterial protein products in yeast. In addition, there are difficulties in NADP regeneration in the yeast engineered ED pathway as NADPH produced in glucose-6-phosphate dehydrogenase reaction, cannot be reoxidized via alcohol dehydrogenase reaction. It has already been reported that the major alcohol dehydrogenases in S. cerevisiae utilize NADH but not NADPH and yeast does not possess NADH/NADPH transhydrogenase (Lescovac et al., 2002; Jeffries and Jin, 2004). In order to maintain a low level of ATP during yeast alcoholic fermentation through EMP pathway, it is not necessary to substitute it for a pathway with lower efficiency. A better approach to achieve the above is to keep EMP pathway unchanged and to lower level of ATP by a more specific approaches that rely on the activation of some cytosolic ATPase or via the induction or construction of some kind of futile cycle to dissipate cellular pool of ATP.
In our previous work, we carried out a successful attempt to decrease intracellular ATP level by overexpression of 5′ part of the S. cerevisiae SSB1 gene encoding cytosolic ATPase domain and by the heterologous gene apy encoding apyrase from Escherichia coli (Sibirny et al., 2010). Some of the constructed strains showed a decrease in cellular ATP level during anaerobic, aerobic or semi-anaerobic cultivation and were characterized by a reduction in cellular biomass yield with the corresponding increase in ethanol yield during glucose utilization under anaerobic, aerobic or semi-anaerobic conditions. We suggested that this approach can be useful for the construction of a new generation of industrial strains of S. cerevisiae which are characterized by improved ethanol yield from conventional (glucose, sucrose) and non-conventional (lignocellulose) feedstocks.
In this application we describe another approach to lowering cellular ATP by overexpression of intact or truncated versions of the S. cerevisiae PHO8 gene, encoding alkaline phosphatase.